Wetware advances: Biological logic gate built by splitting viral gene

Scientists make a genetic AND gate with the help of a T7 bacteriophage.

In recent years, researchers in the messy world of biology have been able to build systems that function like the clean, binary switches on computer chips—and we've covered a number of reports in this area. Unfortunately, most of these share a significant limitation: they rely on proteins from bacteria that act as switches to turn genes on and off under specific conditions. We know about only a limited number of these genetic switches, which may set a severe limit on the number of logical operations we can string together inside a cell.

A paper in this week's PNAS describes a system that may allow us to get around this limitation. The new method takes a protein from a virus that infects bacteria and cuts it in two, making a pair of genes (A and B) that each produce part of the mature protein. The two parts then act as a biological version of an AND logic gate, with output (in the form of protein activity) present only when both A and B interact. When either or both A and B are missing, the output is off.

In biological terms, the inputs usually involve a simple molecule that can be sensed by proteins inside a bacteria. This paper, for example, used two kinds of sugars (arabinose and lactose). When the sugars are present, they attach to proteins inside the cell, activating genes that are controlled by those proteins. To make an AND gate, you need to design a bit of biology that can respond to both of these signals—it should be active only when both a gene regulated by arabinose and a gene regulated by lactose are each active.

This has been done in a variety of ways in the past, but the authors of the new paper (both faculty at Rice University) come up with a clever scheme for doing so. It's clever in part because it's so remarkably simple.

The uses of a T7 bacteriophage

The new work relies on a gene from a virus called the T7 bacteriophage that infects bacteria. Instead of relying on host proteins to ensure that its genes are turned into RNA (and thus into proteins), T7 carries its own gene for a protein that transcribes DNA into RNA. This gene, called (wait for it) T7 RNA polymerase, recognizes DNA sequences on the virus, sticks to them, and then starts copying the DNA nearby into RNA. If you move the sequences it recognizes somewhere else—into the bacterial genome, onto a construct you supply—it will start copying there instead.

T7 RNA polymerase therefore makes a good output. When it's active, you can use it to turn on a variety of genes, thus coordinating a significant response to your logic. But how do you get T7 RNA polymerase to respond to the two different inputs required for an AND gate? You break it in two.

Scientists who were studying T7 RNA polymerase had found that, during purification, it would sometimes get cut into two different pieces, one about four times the size of the other. Either of the parts on its own is inactive, but if you put them together they stick, and the resulting aggregate is active (that is, it would bind to the appropriate DNA sequences and start making RNA, albeit at a slightly slower rate than the intact protein).

It turns out this also works if the two parts are encoded by completely separate genes. You can encode the larger T7 RNA polymerase fragment in a gene that responds to arabinose and the smaller fragment in a gene that responds to lactose. A functional T7 RNA polymerase will only be present when both sugars are present, so you've made your biological AND gate.

By putting a fluorescent protein under the control of T7 RNA polymerase, the authors were able to show that this worked as expected. The cells glowed green only when both sugars were added.

That on its own is pretty good, although not so much better than some previous work in the field. The great part of this system is its flexibility. Because T7 RNA polymerase has been studied extensively, researchers have identified a variety of mutations that alter the protein's ability to bond to specific DNA sequences. A single change in the right location can thus switch T7 RNA polymerase from sticking to (for example) a sequence that includes GACG to one that includes the sequence GCAT. Other changes in the T7 RNA polymerase can alter the sequence it recognizes even further.

Instead of relying on different proteins for every logical operation you need to do (which will quickly exhaust your supply of tractable proteins), you can now build up logic using different forms of T7 RNA polymerase, each recognizing a somewhat different sequence. This doesn't necessarily help with alternate logic operations, like NOT, but having a larger array of potential tools can only make designing biological circuitry easier.